The detection of a ligand by a receptor (for example, detection of an hormone, an antigen or a pathogenic agent by an antibody, detection of an antibody in blood by another antibody, or binding of a chemical toxin) is important in the diagnosis of diseases and finding of useful biomolecules. Many rapid test methods for detecting ligands with high selectivity and sensitivity have been developed in the past years. These include radio-activity based immunoassay, chemiluminescence based immunoassay, magnetic based assay, fluorescence or colorimetric analysis based immunoassay. Immunoassays, such as enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), and radioimmunoassay (RIA), are well known for the detection of ligands such as hormones, antigens or antibodies. The basic principle in many of these assays is that a marker-conjugated (for example, an enzyme-, chromogen-, fluorogen-, or radionucleotide-conjugated) antibody permits antigen detection upon antibody binding. In order for this interaction to be detected as a change in color, fluorescent or radioactive complexes, significant numbers of antibodies must be bound to a correspondingly large number of antigen epitopes. The resulting labeled binding complex must be isolated from the labeled marker molecules for detection. Currently, the isolation methods include ammonium sulfate or antibody precipitation, chromatography, solid phase assay, dialysis and active carbon adsorption.
The heterogeneous immunoassay usually includes the isolation procedure to separate the free analytes (or antibodies) from the bound immunocomplexes. Traditionally, the method for immunocomplex isolation includes fractionation precipitation, immunoprecipitation, column chromatography, solid-phase analysis, dialysis, and the absorption of dextran-coated charcoal. Fractionation precipitation uses the salts or organic solvents to fractionally precipitate target proteins. Immunoprecipitation uses antibodies in complex with the target antigens or antibodies to form the insoluble immunocomplex which is further precipitated by centrifugation. Column chromatography uses chromatographic method to sequentially separate analytes and enriches the immunocomplex in the collection tube. In solid-phase immunoassay, ligands are coated on a plate or a bead before interacting with the receptors. Dialysis can separate the unbound small molecules from the large immunocomplexes by using a molecular sieving membrane. All the methods mentioned above have various disadvantages such as having a complex procedure, or being time-consuming and expensive, which limit the application for high throughput assay. Separation of free analytes from large immunocomplexes can also be achieved by using the dextran-coated charcoal, but the traditional use of dextran-coated charcoal in immunoassay is for diagnosis of small molecules (e.g., steroid hormone or insulin) in serum (Clinical Chemistry 25 (1979) 1402-1405). So far, dextran-coated charcoal has not been able to apply to the analysis of large antigens, or antibodies that are specific for large antigens, and this should be further improved.
The essential element of any immuno-based detection is the probe that binds analyte and, as a part of the analytical platform, generates a measurable signal. Peptide probes that specifically bind to biomolecules are developed as probes to detect analytes. Such peptide probes can be selected by using various methods. For example, a peptide that is an epitope or a mimotope fragment of a pathogenic antigen can be used to screen the infected antiserum. In addition, a peptide that specifically interacts with an antigen or an antibody can be screened by using phage display technique or similar screening methods. Peternko and Vodyanoy summarizes the state of art in development of the probes for detection of biological threat agents and focuses on phage display, which is new in the detection area and specializes in development of molecular probes to target various biological structures (Journal of Microbiological Methods 53 (2003) 253-262). Dennis et al. describes the phage displayed peptide (DICLPRWGCLW) that can specifically interact with serum albumin (The Journal of Biological Chemistry 277(38) (2002) 35035-35043). Bessette et al. screens specific binding peptides for human serum albumin, anti-T7 epitope mAb, human C-reactive protein, HIV-1 GP120 and streptavidin by using bacterial display technique (Protein Engineering, Design & Selection 17(10) (2004) 731-739). Lu et al. (Journal of Virological Methods 119 (2004) 51-54) and Tan et al. (Journal of Clinical Virology 34 (2005) 35-41) develop the phage bearing peptides that interact tightly with the immunodominant region of hepatitis B surface antigen (HBsAg), and demonstrate the ELISA-like methods for HBsAg detection by using these peptides. The above disclosure indicates that the specific peptides have the potential to be used as diagnostic agents.
Some rapid test systems incorporate more than one immunology-based technique into the system to improve specificity and/or sensitivity. Immunology-based rapid assays already in existence can be further modified or incorporated into other systems to improve their performance, which obviates the need to create entirely new detection systems. Array based immunoassay techniques for multi-analyte detection produce high-density arrays formed by spatially addressable synthesis of bioactive probes on a 2-dimensional solid support and have greatly enhanced and simplified the process of biological research and development. Proteomics 2006, 6, 1376-1384 discloses a method for manufacturing peptide microarrays for molecular immune diagnostics. However, these array systems utilize immunology-based assay, so they still have the disadvantages mentioned above.
Wu and Tsai (Journal of Biomolecular Screening 11 (7) (2006) 836-843) discloses a rapid transglutaminase assay for high-throughput screening applications on the basis of magnetic dextran-coated charcoal. This article only mentions the screening of transglutaminase activity by detecting the conjugation of casein and dansylcadaverine, but does not teach the concept of antibody or antigen detection by using a specific peptide probe.
Accordingly, there remains a need for a high-throughput screening method for simultaneously detecting more than one target biomolecule in a sample.